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Supplementary Figure 3. KLH-specific immune responses before and after DC vaccination. (A) KLH-specific T cell proliferation was analyzed before the first vaccination and after each DC vaccination during the first vaccination cycle in peripheral blood of vaccinated metastatic melanoma patients. Per time point each dot represents 1 patient. Proliferative response to KLH is given as proliferation index (proliferation with KLH/proliferation without KLH). The two patients not showing a KLH-specific T cell response are shown in grey. (B) KLH-specific IgG, IgA, and IgM antibodies were quantitatively measured after each vaccination cycle in sera of vaccinated patients. Per isotype each dot represents one patient. Best KLH responses per patient are shown.

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood.Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100.Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth.Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

Supplementary Figure 1. Schematic overview of the 44 h mDC vaccine preparation cycle and vaccination strategy.

Supplementary Table 2 PDF file - 30K, Patient characteristics and historical controls

Supplementary Figure 2 PDF file - 46K, Supplementary Figure 2 Plasmacytoid DCs acquire CCL21-driven chemotactic ability upon activation

Supplementary Figure 1 PDF file - 34K, Supplementary Figure 1 CliniMACS-based pDC isolation is feasible

Supplementary Table 1 PDF file - 29K, Induration (mm) of the different injection sites in the DTH skin test

Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2. Citation Format: Anna Baranska, Marc Schuster, Marek Wieczorek, Sebastian Flade, Lennart Wessels, Burhan Karaca, Verena Traska, Mario Assenmacher, Gregor Winkels. Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P23.

In recent years, CAR T cell mediated immunotherapy has shown clinical efficacy in the fight against cancer. Increased research efforts to address several challenges associated with CAR T cell therapy for solid tumor treatment for instance, such as poor efficacy and unwanted side effects, are required to progress current approaches. Fast, efficient and flexible CAR T workflows can offer the opportunity to progress such research. At Miltenyi Biotec we have established an optimized CAR T cell research workflow, combining cell isolation, culture and analysis tailored for CAR T cell generation that can be transferred into clinical setting. Using our StraightFrom® MicroBeads, the fundamental step of isolating high quality T cells as starting material to generate functional CAR T cells, can be performed directly from whole blood, buffy coat and blood products without density gradient centrifugation. Thereby, experimental procedure is accelerated and allows for automation on platforms such as the MultiMACS™ Cell24 Separator Plus and the MultiMACS™ X, whilst maintaining the quality, functionality and downstream compatibility of the isolated T cells, resulting in an ideal starting population for the generation of fully functional CAR T cells. In conclusion, our innovative tools can reduce experimental time and increase the reproducibility and quality of data obtained in your CAR T cell research efforts in immuno-oncology and immunotherapy.

Aim: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. Patients & methods: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. Results: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. Conclusion: This method was feasible and safe but was minimally immunogenic.

Enrichment of leukocytes from blood products like buffy coat, leukocyte reduction chambers (LRSC) or leukapheresis material can be time consuming and could influence the physiology of the target cells. Our new StraightFrom cell separation reagents were specifically developed to minimize time and handling steps, omitting the need to perform density gradient centrifugation methods or erythrocyte lysis prior to isolation thereby preserving cell intergrity. Based on positive isolation, Th cells (CD4), cytotoxic T cells (CD8), Monocytes (CD14), B cells (CD19) or NK cells (CD56) can be isolated from complete buffy coats, LRSC or half pack LPs within less than 30 minutes with purities above 95%. The positive enrichment process enables thereby the consecutive enrichment of more than one cell population from a sample and is automatable. Using various assays, we show that functionality (e.g activation and expansion, cytokine secretion, ROS production) and viability of the target cells are preserved without cell activation. Ultimately, this optimized magnetic cell separation technology allows the isolation of physiologically relevant cells in a quick and easy manner.

Plasmacytoid dendritic cells (PDC) are the main type I interferon (IFN-I) producers and play a central role in innate and adaptive immunity. CD303 (BDCA-2) is a type II c-type lectin specifically expressed by human PDC. CD303 signaling induces tyrosine phosphorylation and Src kinase dependent calcium influx. Cross-linking CD303 results in the inhibition of IFN-I production in stimulated PDC. Here, we demonstrate that PDC express a signalosome similar to the BCR signalosome, consisting of Lyn, Syk, Btk, Slp65 (Blnk) and PLCgamma2. CD303 associates with the signaling adapter FcR gamma-chain. Triggering CD303 leads to tyrosine phosphorylation of Syk, Slp65, PLCgamma2 and cytoskeletal proteins. Analogous to BCR signaling, CD303 signaling is likely linked with its internalization by clathrin-mediated endocytosis. Furthermore, CD303 signaling leads to reduced levels of transcripts for IFN-I genes and IFN-I-responsive genes, indicating that the inhibition of IFN-I production by stimulated PDC is at least partially regulated at the transcriptional level. These results support a possible therapeutic value of an anti-CD303 mAb strategy, since the production of IFN-I by PDC is considered to be a major pathophysiological factor in systemic lupus erythematosus patients.

Purpose: Thus far, dendritic cell (DC)-based immunotherapy of cancer was primarily based on in vitro–generated monocyte-derived DCs, which require extensive in vitro manipulation. Here, we report on a clinical study exploiting primary CD1c+ myeloid DCs, naturally circulating in the blood. Experimental Design: Fourteen stage IV melanoma patients, without previous systemic treatment for metastatic disease, received autologous CD1c+ myeloid DCs, activated by only brief (16 hours) ex vivo culture and loaded with tumor-associated antigens of tyrosinase and gp100. Results: Our results show that therapeutic vaccination against melanoma with small amounts (3–10 × 106) of myeloid DCs is feasible and without substantial toxicity. Four of 14 patients showed long-term progression-free survival (12–35 months), which directly correlated with the development of multifunctional CD8+ T-cell responses in three of these patients. In particular, high CD107a expression, indicative for cytolytic activity, and IFNγ as well as TNFα and CCL4 production was observed. Apparently, these T-cell responses are essential to induce tumor regression and promote long-term survival by stalling tumor growth. Conclusions: We show that vaccination of metastatic melanoma patients with primary myeloid DCs is feasible and safe and results in induction of effective antitumor immune responses that coincide with improved progression-free survival. Clin Cancer Res; 22(9); 2155–66. ©2015 AACR.

We have recently described a panel of monoclonal antibodies (mAb), that recognize two novel leukocyte surface antigens, BDCA-2 and BDCA-4. BDCA-2 is a novel type II C-type lectin specifically expressed by plasmacytoid dendritic cells (PDCs) that can internalize antigen for presentation to T cells. Furthermore, signaling via BDCA-2 may play a role in switching from interferon (IFN)-alpha/beta-controlled to interleukin (IL)-12-controlled immune response pathways, as triggering of BDCA-2 potently inhibits secretion of IFN-alpha/beta by PDCs and thereby promotes IL-12 p70 production in PDCs and other cells. Viruses may exploit this switch to escape innate antiviral immunity, but it may be beneficial for patients with systemic lupus erythematosus (SLE) if induced, for instance by anti BDCA-2 mAb treatment. BDCA-4 is shown here to be identical to neuropilin-1 (NP-1), a neuronal receptor for the axon guidance factors belonging to the class-3 semaphorin subfamily, and a receptor on endothelial and tumor cells for vascular endothelial growth factor (VEGF-A). In blood and bone marrow, BDCA-4/NP-1 is exclusively expressed on PDCs, but in tonsils also on a few other cells, primarily follicular B helper memory T cells (T(FH)).

Contains fulltext : 119153.pdf (Publisher’s version ) (Open Access) Vaccination against cancer by using dendritic cells has for more than a decade been based on dendritic cells generated ex vivo from monocytes or CD34(+) progenitors. Here, we report on the first clinical study of therapeutic vaccination against cancer using naturally occurring plasmacytoid dendritic cells (pDC). Fifteen patients with metastatic melanoma received intranodal injections of pDCs activated and loaded with tumor antigen-associated peptides ex vivo. In vivo imaging showed that administered pDCs migrated and distributed over multiple lymph nodes. Several patients mounted antivaccine CD4(+) and CD8(+) T-cell responses. Despite the limited number of administered pDCs, an IFN signature was observed after each vaccination. These results indicate that vaccination with naturally occurring pDC is feasible with minimal toxicity and that in patients with metastatic melanoma, it induces favorable immune responses. Cancer Res; 73(3); 1063-75. (c)2012 AACR.

Aim Serological crossmatch analysis (i.e. complement-dependent cytotoxicity (CDC)) is commonly done before solid organ transplantation to detect donor specific antibodies that may lead to graft rejection or dysfunction. Routine analysis is often performed on isolated donor lymphocyte subpopulations together with recipient serum. Enrichment of cells for CDC can be exceedingly time consuming or results in co-enrichment of non-target or dead cells. We aimed to develop a fast and convenient method for enrichment of donor lymphocyte subsets directly from whole blood. Methods Using MACSxpress technology, untouched human leukocyte subsets can be isolated within only 20 min from up to 30 ml of anticoagulated whole blood. For the enrichment of B, T or a mixture of BT cells whole blood or cell suspensions obtained from rinsed donor spleens were incubated with the respective bead cocktails for 5 min and the tube placed for 15 min in the magnetic field of a separator. With the tube inside the magnetic field, the supernatant, containing the enriched target cells, was collected. Magnetically labeled non-target cells as well as aggregated erythrocytes are retained in the tube. Purity and viability of the enriched cell populations were determined by flow cytometry and functionality was shown in CDC assays. Results MACSxpress-enriched lymphocyte subsets (B/T/BT cells) from whole blood had average purities of 87/96/98% with yields of 60/54/38% ( n = 27/29/23). Cell enrichment from spleen single cell suspensions resulted in purities of 97/96/98% and recoveries of 27/32/29% ( n = 5) (B/T/BT cells). CDC analysis of positive and negative control sera with enriched cells showed no influence of fluorescent readout by the enrichment process. Conclusion Using MACSprep HLA Isolation Kits, lymphocytes for crossmatch analysis can be enriched within 20 min from whole blood without the need of expensive lab equipment or preparation of PBMC by density gradient centrifugation. G. Winkels: Employee; Company/Organization; Miltenyi Biotec.

CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8+ T-cells. Two major MDSC subsets were recently shown to play an equal role in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b+/Gr-1high, CD11b+/Gr-1int, and CD11b+/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b+/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8+ T-cell activation, while CD11b+/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b+/Gr-1int but not CD11b+/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b+/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b+/Gr-1int and CD11b+/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumor-induced tolerance whereas G-CSF, which preferentially expanded CD11b+/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte-macrophage progenitors in the bone marrow inducing local expansion of CD11b+/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.

The dendritic cell (DC) immunoreceptors (DCIR) and DC-immunoactivating receptors (DCAR) represent a subfamily of cell surface C-type lectin receptors (CLR), whose multifunctional capacities range from classical Ag uptake and immunoregulatory mechanisms to the involvement in DC ontogeny. On the basis of the generation of specific mAbs, we functionally characterized mouse DCAR1 (mDCAR1) as a member of the DCIR/DCAR family. Expression of mDCAR1 was strongly tissue dependent. mDCAR1 expression on DCs was restricted to the CD8+ DC subset in spleen and thymus and on subpopulations of CD11b+ myeloid cells in bone marrow and spleen, whereas the molecule was not detectable on both cell types in lymph nodes and peripheral blood. With respect to the function of CLRs as pattern recognition receptors, Ag delivered via mDCAR1 was internalized, was trafficked to early and late endosomes/lysosomes and, as a consequence, induced cellular and humoral responses in vivo even in the absence of CD40 stimulation. Intriguingly, upon triggering mDCAR1, CD8+ DCs increased the secretion of bioactive IL-12, whereas IL-10 release is markedly reduced, thereby indicating that Ag recognized by mDCAR1 induces enhanced proinflammatory responses. These data indicate that mDCAR1 is a functional receptor on cells of the immune system and provides further insights into the regulation of immune responses by CLRs.

C-type lectins (CLEC) comprise a superfamily of cell surface proteins, whose multifunctional capacity has been shown to address both, innate and adaptive immune response. Here, we describe mDCAR1 a...

Contains fulltext : 49700.pdf (Publisher’s version ) (Open Access) Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immune responses by producing type I interferons. Although human pDCs can induce T cell responses upon viral infection, it remains unclear if pDCs can present exogenous antigens. Here, we show that human pDCs exploit FcgammaRII (CD32) to internalize antigen-antibody complexes, resulting in the presentation of exogenous antigen to T cells. pDCs isolated from melanoma patients vaccinated with autologous monocyte-derived peptide- and keyhold limpet hemocyanin (KLH)-loaded dendritic cells, but not from nonvaccinated patients or patients that lack a humoral response against KLH, were able to stimulate KLH-specific T cell proliferation. Interestingly, we observed that internalization of KLH by pDCs depended on the presence of serum from vaccinated patients that developed an anti-KLH antibody response. Anti-CD32 antibodies inhibited antigen uptake and presentation, demonstrating that circulating anti-KLH antibodies binding to CD32 mediate KLH internalization. We conclude that CD32 is an antigen uptake receptor on pDCs and that antigen presentation by pDCs is of particular relevance when circulating antibodies are present. Antigen presentation by pDCs may thus modulate the strength and quality of the secondary phase of an immune response.

Dendritic cells (DC) can either be generated from progenitors such as stem cells or CD14+ monocytes, or isolated directly from the blood. Blood-derived DC are present as at least two distinct populations-myeloid and plasmacytoid DC. Here we describe methods for the clinical-grade isolation of blood DC and DC precursors using the CliniMACS. We describe the isolation of ultra-pure monocytes in order to generate large numbers of monocyte-derived DC, and also new methods for the direct isolation of blood DC. Isolation of blood DC in large numbers means that natural DC with different properties can be investigated for their clinical function for the first time.

Dendritic cells (DC) can either be generated from progenitors such as stem cells or CD14+ monocytes, or isolated directly from the blood. Blood-derived DC are present as at least two distinct populations-myeloid and plasmacytoid DC. Here we describe methods for the clinical-grade isolation of blood DC and DC precursors using the CliniMACS. We describe the isolation of ultra-pure monocytes in order to generate large numbers of monocyte-derived DC, and also new methods for the direct isolation of blood DC. Isolation of blood DC in large numbers means that natural DC with different properties can be investigated for their clinical function for the first time.

CD303 is a calcium dependent type II lectin also known as Blood-Dendritic-Cell-Antigen-2 (BDCA-2) specifically expressed by human plasmacytoid dendritic cells (PDC). We have previously shown that monoclonal antibody (mAb) ligation of CD303 induces mAb endocytosis, calcium mobilization, protein tyrosine phosphorylation and inhibition of type I interferon (IFN I) production in stimulated PDC. Here we show that CD303 signaling and internalization in many aspects resembles B cell receptor (BCR) signaling and internalization. Instead of CD79a and CD79b, CD303 appears to use the Fc-receptor-common-gamma-chain as transmembrane adaptor protein. Like BCR signaling, CD303 triggering leads to SYK and BLNK phosphorylation. Signal transduction most likely involves activation of the phospholipase C-gamma 2, the phosphoinositide-3 kinase and the protein kinase C delta. Therefore, inhibition of IFN I production in stimulated PDC can be mimicked by PMA. Western blotting and peptide mass fingerprinting show that tyrosine phosphorylation occurs at cytoskeletal proteins (actin, alpha/beta-tubulin, profilin, alpha-actinin), proteasome activator subunit and the clathrin heavy chain, indicating clathrin-mediated endocytosis and vesicle trafficking. Finally, CD303 triggering inhibits TNF-alpha and CpG ODN-induced NF-kappa B activation in PDC.